*2.2. Flow Cytometry*

Bacterial viability in samples was measured by flow cytometry using the LIVE/DEAD BacLight ™ Bacterial Viability and Counting Kit (L34856, Molecular Probes) according to manufacturer instructions (Molecular Probes) [20]. Briefly, 977 μL of 0.9% NaCl, 1.5 μL of SYTO9, 1.5 μL of propidium iodide (PI) and 10 μL of diluted sample were added to a flow cytometry analysis tube. Samples were 10-fold diluted in 0.9% NaCl. The tube was incubated for 15 min in a dark at room temperature. A quantity of 10 μL of the microsphere suspension (beads) was added to the stained sample. The total volume of the sample in the flow cytometry analysis tube was 1000 μL. The samples were analyzed on a LSR Fortessa flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) with FACS Diva v8 software (Becton Dickinson). The gating strategy is shown in Figure 1 and shows three main cell populations—alive, dead and unknown (probably alive, probably dead) with a special "double negative" group of cells (SYTO9−PI−). The number of bacteria per mL in each analyzed gate was counted according to the following formula taken from the manufacturer materials:

> ((# *o f events* ∈ *gatedbacteriaregion*) × (*dillution f actors*)) [(# *o f events* ∈ *beadregion*) × <sup>10</sup>−<sup>6</sup>] = *bacteria*/mL

**Figure 1.** Gating strategy shown on one of the samples. SYTO9-positive PI-negative cells were considered alive, SYTO9-negative PI-positive cells were considered dead, other cells were considered as unknown, with special gating on SYTO9-negative PI-negative cells, which were called "double negative" cells.

#### *2.3. Cultivation of Stool Microbiota*

Samples were plated on six di fferent agar media and incubated under conditions as follows. (i) CNA medium (colistin nalidixic acid agar; Oxoid, Basingstoke, UK) for cultivation of Gram-positive aerobes, an enriched agar medium containing sheep's blood, colistin and nalidixic acid (to inhibit the growth of Gram-negative bacteria). Culture conditions: aerobic with 5% CO2, 37 ◦C, 48 h. (ii) MacConkey medium (bioMérieux, Marcy l'Etoile, France) for the isolation of Gram-negative rods, containing bile salts and crystal violet (to inhibit the growth of Gram-positive bacteria). Culture conditions: aerobic, 37 ◦C, 48 h. (iii) Bile and esculin (CC) medium (Oxoid)—a medium intended for the isolation and identification of bacteria belonging to the genus *Enterococcus*, which grow well in the presence of bile and have the ability to break down esculin. Culture conditions: aerobic, 37 ◦C, 48 h. (iv) Schaedler Anaerobe KV Selective Agar with freeze-dried horse blood and the addition of kanamycin and vancomycin (bioMérieux)—a highly nutritious medium for the selective growth and isolation of anaerobic bacteria, especially of the genus *Bacteroides* and *Prevotella*. Culture conditions: anaerobic, 37 ◦C, 4 days. (v) Schaedler Anaerobe KV Selective Agar with freeze-dried horse blood (bioMérieux)—a highly nutritious medium for the isolation of absolute and relative anaerobes. Culture conditions: anaerobic, 37 ◦C, 4 days. (vi) Sabouraud agar with gentamicin and chloramphenicol (Oxoid)—selective medium for cultivation of mold and yeast, high glucose concentration. The presence of antibiotics (chloramphenicol and gentamicin) and acidic pH inhibits bacterial growth; the presence of antibiotics is another selection factor. Culture conditions: aerobic, 37 ◦C, 10 days. The anaerobic incubations were carried out in anaerobic jars and atmosphere generators (Oxoid).

After the initial sample processing, colonies were selected (at least one colony per morphology) for identification using a Microflex LT mass and MBT Compass IVD Biotyper software (Bruker Daltonics, Bremen, Germany). The colonies were deposited on a MALDI-TOF (Bruker Daltonics) target microflex and extracted with 5% formic acid, air dried and then overlaid with 1 μL matrix solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid. Two spots were examined for each colony. The Biotyper software was used to compare the protein profile of the cultured bacteria from a database of Bruker consisting of 2750 of protein profiles. A score >1.9 was considered a high-level identification of a species, a score >1.7 indicated the identification of a genus. Strains of bacteria with scores lower than 1.7 were considered unidentified.

To enumerate the number of colony-forming units (CFU) in the stool samples, 0.2 g of stool was diluted in 1 mL of phosphate-bu ffered saline (PBS), and 1–5 μL of watery sample was spread on each media. Bacterial counts were recorded as CFU per gram of feces for each isolated species.

### *2.4. DNA Sequencing*

Total bacterial DNA was extracted using a Qiagen DNeasy Power Soil kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and stored at −20 ◦C. Using isolated

DNA as a matrix, PCR reactions were performed in triplicate (to reduce PCR bias) using a Bakt\_341F 5-CCTACGGGNGGCWGCAG-3 and Bakt\_805R 5-GACTACHVGGGTATCTAATCC-3 primer pair amplifying the variable V3 and V4 regions of the 16S rRNA genes [21,22]. Electrophoretic analysis was performed for each of three replicates for qualitative and quantitative evaluation of the PCR products. Then, products of three independent PCR reactions for each sample were mixed and used for the DNA sequencing as one amplicon to minimize the error due to the selectivity of the PCR reactions. The amplified PCR products were sequenced using an Illumina MiSeq instrument (Illumina, San Diego, CA, USA) in paired-end mode using a v3 chemistry kit (Illumina) at BIOBANK LAB (Chair and Department of Molecular Biophysics, University of Lodz, Łód ´z, Poland).
